JP4227591B2 - Solution composition for sequencing reactant cleanup - Google Patents

Description

(background)
ABI PRISM® BigDye® terminator available from Applied Biosystems, Inc. v. Commercially available DNA sequencing kits, such as 1.0, 1.1, 2.0, 3.0 and 3.1 Ready Reaction Cycle Sequencing Kits, are fluorescently labeled. A molecule or dye terminator is used as a component. For example, a dye based terminator, deoxynucleoside triphosphate, sequencing enzyme, magnesium chloride and buffer premixed, and a fluorescence based cycle sequence for single or double stranded DNA templates and polymerase chain reaction fragments Suitable for performing a decision reaction.

  Since dye terminators are not natural substrates of DNA polymerases, generally high concentrations must be provided relative to the natural dNTP substrate to ensure their incorporation into sequencing products during polymerization. However, unincorporated fluorescently labeled molecules are difficult to remove when present at high concentrations. If not properly removed, unincorporated fluorescently labeled molecules can interfere with downstream analysis (eg, DNA sequencing), such as by co-migration with short sequencing products in electrophoresis. In fact, these molecules have a tendency to form insoluble complexes at high concentrations. They are particularly problematic in reactions that utilize high concentrations of sequencing chemistry (eg, 1 ×, 1/2 ×, and 1/4 × intensity reactions).

  Conventional methods for removing unincorporated dye terminators from sequencing reactions prior to electrophoresis include alcohol precipitation and gel filtration. However, the salt competes with sequencing products for electrokinetic injection into the capillary sequencing device and must still be removed. Since ethanol precipitation has insufficient salt removal capacity and the efficiency of electrokinetic injection of sequencing products is inversely proportional to salt concentration, its use as a method for preparing samples prior to capillary electrophoresis Unlikely. Gel filtration is a centrifugation-based method that is difficult to automate, which is important for high-throughput DNA sequencing.

  Another method for removing unincorporated fluorescently labeled molecules such as dye terminators is MultiScreen® or Montage (trade name) commercially available from Millipore Corporation. Use of 384-SEQ ultrafiltration plates is included. These plates are fully automatable and provide a cost and time efficient method to replace conventional ethanol precipitation for dye terminator removal. They work by vacuum filtration, thus eliminating the need for centrifugation, ethanol drying steps and manifold degradation routines. Solvents such as formamide or EDTA help to solubilize the dye terminator and prevent aggregate formation. The sequencing product is purified by filtering to dryness and then washing away the salt and dye terminator. The purified sequencing product is then resuspended from the membrane surface and ready for introduction into a DNA sequencer.

However, the introduction of new sequencing chemistry by various manufacturers continues to present purification challenges. In addition, current ultrafiltration techniques allow for substantially purified DNA sequencing with a reaction intensity of 1 microliter sequencing reagent (1/8 ^th BDT v3.0) per DNA sequencing reaction. Although providing a product, at higher concentrations, artifacts known as dye blobs are evident on the electropherogram. These artifacts are generally visible when using other clean-up methods such as gel filtration and alcohol precipitation, despite special modifications to the protocol recommended by the manufacturer to eliminate them. Is.

  Accordingly, it would be desirable to provide a cost-effective and efficient solution for reducing or eliminating unincorporated fluorescently labeled molecules and residual salts from sequencing reactions.

(Overview)
The problems of the prior art have been overcome by the present invention which provides wash solutions and methods for purifying sequencing reaction products. The wash solution preferably comprises about 1 mM to about 60 mM guanidine in a low ionic solution. In that method aspect, the invention includes adding a wash solution to the sequencing reaction product prior to filtration and then filtering to reduce or eliminate unincorporated dye terminators. The purified sequencing product can then be resuspended and transferred to an appropriate substrate for sequencing or further preparation. Dye spots formed from unincorporated dye terminators no longer interfere with the resolution of the electropherogram produced by sample electrophoresis.

(Detailed explanation)
We have found that effective amounts of guanidine in sequencing reaction products break up aggregates and / or prevent their formation, as demonstrated in the electropherogram, even at higher reaction scales. I found out. An effective amount of guanidine is an amount sufficient to reduce the presence of unincorporated dye terminators to such an extent that they do not adversely interfere with downstream analysis, particularly electrophoresis of sequencing reaction products. Harmful interference to electrophoresis is manifested during the appearance of pigment spots in the electropherogram, and the presence of pigment spots makes it difficult or impossible to accurately separate the sequencing products. Such interference is particularly noticeable with shorter DNA. Higher concentrations of dye terminators also interfere with sequencing further downstream.

  Excess guanidine may result in unacceptable loss of shorter sequencing products. Insufficient amounts of guanidine can lead to an inadequate reduction of harmful artifacts, or their reappearance. Suitable amounts of guanidine are in the range of about 1 to about 60 mM, more preferably about 1 to about 30 mM, with about 5 to about 10 mM being particularly preferred. It is surprising that only a relatively low concentration of guanidine was effective in sufficiently reducing or eliminating pigment spots from the electropherogram.

  Preferably, guanidine is used in the form of a salt. Suitable salts include guanidine chaotropes such as guanidine carbonate or guanidine hydrochloride. The particular salt used should be selected to avoid undesired interactions during the reaction. Preferably, the guanidine salt is used in a low ionic solution such as 0.3 mM EDTA having a pH of about 5-11, preferably 8-10.

  In an aspect of the method, the invention provides a step of providing a defined amount of sequencing reaction product, a step of preparing at least one ultrafiltration membrane having at least one surface, and a surface of the ultrafiltration membrane. A step of introducing a sequencing reaction product, a step of adding a guanidine washing solution either before or after the sequencing reaction product is introduced to the surface of the ultrafiltration membrane, and a sequence determination substantially free of contaminants Applying a constant pressure difference or a driving force such as a vacuum to the surface of the membrane sufficient to produce a reaction product. The addition of guanidine cleaning solution and application of driving force can be repeated. Sequencing reaction products are retained on the membrane surface, while contaminants containing dye terminators are discarded by filtration. Preferably, the sequencing reaction product is filtered to dryness (eg, until no visible liquid remains) and the driving force is maintained for about 15 seconds after filtering to dryness (in a multiwell device). If multiple filtrations are performed simultaneously, the driving force is preferably maintained until the last well is empty). The sequencing reaction product is washed (such as with the washing solution of the present invention), filtered to dryness again, resuspended in a low ionic solution known to those skilled in the art, such as formamide or water, and sequenced ( For example, electrokinetic injection), restriction digestion or transfer to a suitable substrate for further preparation (eg by pipetting).

  The table below illustrates appropriate amounts of ingredients for various concentrations of sequencing chemistry using the ABI BigDye® terminator kit.

  Suitable ultrafiltration membranes have a cutoff molecular weight of about 1000 to 30,000 daltons, preferably about 3,000 to 15,000 daltons. They can be made from a variety of materials including, but not limited to, polyamides, polysulfones, polyether sulfones, polyaryl sulfones, cellulose derivatives, regenerated cellulose, and polyvinylidene fluoride. They may be symmetric or asymmetric, but the latter is preferred. For high throughput applications, preferably multiple ultrafiltrations are performed simultaneously, most preferably using a 384 well filter plate. Other sized plates including a 96 hole format can also be used. Suitable filter plates include MultiScreen® or MONAGE® (trade name) 384-SEQ filter plates, commercially available from Millipore.

  A suitable vacuum is sufficient to reduce or eliminate the dye terminator and is generally in the range of about 15 to about 28 inches of mercury, preferably about 23 to 25 inches of mercury. In general, the driving force is applied slightly (about 15 seconds) longer than the time required to remove all visible liquid from the well, which is typically about 3-4 minutes.

Sequencing reactions were set up with thermal cycling plates (96 wells) and amplified using an appropriate thermal cycling program. The reaction components were 5 pmol M13 primer, 200 ng pLH2 (template), 8 μl big die premix and enough Milli-Q® water to make a final volume of 20 μl. All mixtures were transferred to montage SEQ ₉₆ plates commercially available from Millipore. The plate was placed on a vacuum manifold and a 23-25 inch Hg vacuum was applied for 3-4 minutes until no visible liquid remained in the wells. The vacuum was continued for about 15 seconds and the plate was removed from the manifold. Excess liquid was removed from the bottom of the plate by simply pressing the plate against the stacked paper towels. The plate was returned to the manifold and 25 μl of solution (no guanidine) was added. Again, a 23-25 inch Hg vacuum was applied for 3-4 minutes until no visible liquid remained in the wells, and for about 15 seconds thereafter. Excess liquid was removed from the bottom of the plate by removing the plate from the manifold and again pressing the plate against the stacked paper towels. The DNA was resuspended by adding 25 μl of the injection solution and pipetting up and down 15-20 times. Purified sequencing product was injected into the ABI 3700 sequencer at 2 KV for 15 seconds. The results are shown in FIGS. 1A and 1B. Large pigment spots are evident.

Sequencing reactions were set up with thermal cycling plates (96 wells) and amplified using an appropriate thermal cycling program. The reaction components were 5 pmol M13 primer, 200 ng pLH2 (template), 8 μl big die premix and enough Milli-Q® water to make a final volume of 20 μl. 30 μl of wash solution (no guanidine) was added to each sequencing reaction. After thermal cycling, 30 μl of a sequencing wash solution containing 0.5 mM guanidine in pH 8 0.3 mM EDTA was added to each reaction and mixed gently. All mixtures were transferred to montage SEQ ₉₆ plates commercially available from Millipore. The plate was placed on a vacuum manifold and a 23-25 inch Hg vacuum was applied for 3-4 minutes until no visible liquid remained in the wells. The vacuum was continued for about 15 seconds and the plate was removed from the manifold. Excess liquid was removed from the bottom of the plate by simply pressing the plate against the stacked paper towels. The plate was returned to the manifold and 25 μl of sequencing wash solution was added. Again, a 23-25 inch Hg vacuum was applied for 3-4 minutes until no visible liquid remained in the wells, and for about 15 seconds thereafter. Excess liquid was removed from the bottom of the plate by removing the plate from the manifold and again pressing the plate against the stacked paper towels. The DNA was resuspended by adding 25 μl of the injection solution and pipetting up and down 15-20 times. The purified sequencing product was injected into the ABI 3700 sequencer at 2 kV for 15 seconds. The results are shown in FIGS. 2A and 2B. The reduction in pigment spots as compared to FIGS. 1A and 1B is evident.

  The same procedure as in Example 1 was performed except that only 4 μl of big die premix was used and the final volume was half the reaction scale of 10 μl. The results are shown in FIGS. 3A and 3B.

  The same procedure as in Example 2 was performed except that only 4 μl of big die premix was used and the final volume was half the reaction scale of 10 μl. The results are shown in FIGS. 3A and 3B. Compared to FIGS. 3A and 3B, the lack of pigmented spots is evident.

  The same procedure was performed as in Example 1 except that only 2 μl of big die premix was used and the reaction volume was a quarter of a final volume of 5 μl. Injection into the ABI 3100 Avant sequencer was performed at 1 kV for 22 seconds. The results are shown in FIGS. 5A and 5B.

  The same procedure as in Example 2 was performed except that only 2 μl of big die premix was used and the final volume was half the reaction scale of 5 μl. Injection into the ABI 3100 Avant sequencer was performed at 1 kV for 22 seconds. The results are shown in FIGS. 6A and 6B. Compared to FIGS. 5A and 5B, the lack of pigmented spots is evident.

1 is an electropherogram of a full scale reaction according to the prior art. FIG. 1B is a raw trace of the reactive pigment spot of FIG. 1A. 2 is an electropherogram of a full scale reaction according to the present invention. FIG. 2B is a raw trace of the reduced pigment spot of FIG. 2A. FIG. 2 is an electropherogram of a half scale reaction according to the prior art. 3B is a raw trace of the reactive pigment spot of FIG. 3A. 2 is an electropherogram of a half scale reaction according to the present invention. FIG. 4B is a raw trace showing the lack of pigmented spots in FIG. 4A. FIG. 2 is an electropherogram of a quarter scale reaction according to the prior art. 5B is a raw trace showing reactive pigment spots in FIG. 5A. FIG. 2 is an electropherogram of a quarter scale reaction according to the present invention. FIG. 6B is a raw trace showing the lack of pigmented spots in FIG. 6A.

Claims (6)

By removing the dye terminator unincorporated from sequencing reactions produced product, a method of purifying the reaction product for sequencing,
Providing sequencing reaction products containing unincorporated dye terminator contaminants ;
Providing at least one ultrafiltration membrane having at least one surface;
To remove the dye terminators contaminants unincorporated from the sequencing for the reaction producing compound, to prepare a solution containing guanidine 0.5 to 10 mm;
Introducing the sequencing reaction product and the solution onto the at least one surface of the ultrafiltration membrane;
Producing a purified sequencing reaction product by applying a driving force to the ultrafiltration membrane to remove unincorporated dye terminators .   The method of claim 1, further comprising resuspending the purified sequencing reaction product in a low ionic solution. 3. The method of claim 2, further comprising transferring the resuspended sequencing reaction product to a sequencing substrate. By removing the dye terminator unincorporated from sequencing reactions produced product, a method of purifying the reaction product for sequencing,
Providing sequencing reaction products containing unincorporated dye terminator contaminants ;
Providing a plurality of wells each having an ultrafiltration membrane having at least one surface;
To remove the dye terminators contaminants unincorporated from the sequencing for the reaction producing compound, to prepare a solution containing guanidine 0.5-5 mM;
Introducing the sequencing reaction product and the solution into the plurality of wells;
Producing a purified sequencing reaction product by applying a driving force to each of the plurality of wells to remove unincorporated dye terminators .   5. The method of claim 4, further comprising resuspending the purified sequencing reaction product in a low ionic solution. 6. The method of claim 5, further comprising transferring the resuspended sequencing reaction product to a sequencing substrate.

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